Lipids present in blood are incorporated in the structure of lipoprotein, except for free fatty acid bound with albumin, and are present as chylomicron (CHM), very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL), and the like. Cholesterol therein is particularly distributed in VLDL, LDL, and HDL. HDL is allegedly a preventive factor for heart diseases attributed to arteriosclerosis. Thus, the measurement of high density lipoprotein cholesterol (HDL-C) instead of HDL clinically has a more important meaning. Ultracentrifugation, electrophoresis, and precipitation methods are widely known as current methods for measuring HDL-cholesterol.
The ultracentrifugation method is not suitable for daily examination for such reasons that: it requires a long time for separation procedures; and inexpensive measurement cannot be expected because an apparatus itself is expensive. The electrophoresis method still has a problem in light of quantification for such reasons that: its separation ability differs depending on a difference in electrophoresis support medium; and the method differs depending on use conditions and detection reagents used. Thus, the precipitation method is widely used as current daily examination.
The precipitation method is a method comprising using, for example, a combination of a polyanion and a divalent metal ion, as a precipitating reagent to precipitate CHM, LDL, and VLDL and measuring cholesterol in HDL, that is, HDL-cholesterol, remaining in the supernatant by use of a chemical reagent or enzyme. A combination of a sulfated polysaccharide and an alkaline earth metal ion or a divalent metal ion other than alkaline earth, an inorganic polyanion salt, polyethylene glycol, and so on, which have been well known since the early 1960s by T. Nakai, “HDL-Metabolism, Assay Methods and Clinical Application” (Chugaiigaku Co., Ltd., 1986) and other various documents and textbooks, are widely used as the precipitating reagent. Specific examples of the precipitating reagent include a heparin-calcium reagent, a dextran sulfate-magnesium reagent, and a phosphotungstic acid-magnesium reagent.
The precipitation method is a method in which serum is mixed with the precipitating reagent and left for a certain period of time, and after centrifugation at approximately 3000 revolutions per minute, an aliquot of the supernatant portion is separated and subjected to chemical reaction or enzyme reaction to quantify HDL-cholesterol.
The precipitation method presents problems derived from the precipitating reagent and problems derived from centrifugation procedures. Therefore, JP Patent Publication (Kokai) Nos. 55-78254A (1980), 55-93065A (1980), 61-263467A (1986), and 62-19768A (1987), JP Patent Publication (Kokoku) No. 1-39553B (1989), etc. have described various methods for improving precipitating agents for enhancing precipitation efficiency.
The major disadvantage of the conventional precipitation method is that the use of a reagent rich in triglyceride sometimes results in the partial floating of precipitates after centrifugation. Therefore, the precipitation method presents such a big problem that the adjustment of centrifugation conditions is required. In a method using phosphotungstate-magnesium ions, precipitation sometimes varies depending on the pH of the solution. Therefore, the method presents such a problem that the strict adjustment of pH is required.
When a centrifuged supernatant, particularly a small amount of the supernatant, is separated, the boundary region between precipitates and the supernatant is difficult to determine by visual observation. Therefore, quantitative analysis precision is sometimes lowered due to reproducibility and precision problems and variations among individuals. It has been demanded to improve these disadvantages attributed to centrifugation procedures.
A direct method that does not require these complicated procedures and is available for an automatic analyzer has spread rapidly in recent years. For example, JP Patent Publication (Kokai) No. 8-131197A (1996) has disclosed a method comprising sufficiently reacting sulfated cyclodextrin used as an aggregating agent with lipoprotein other than HDL, then allowing an enzyme modified with polyoxyethylene glycol to act thereon, and measuring cholesterol in HDL. WO98/26090 has disclosed a method comprising a first step of eliminating lipoprotein other than HDL by use of catalase and a second step of measuring HDL-C by use of an activator that specifically acts on HDL. Furthermore, JP Patent Publication (Kokai) No. 9-96637A (1997) has described a method comprising initially allowing an antibody against lipoprotein other than HDL to act thereon, subsequently dissolving HDL, and measuring cholesterol in HDL.
However, these methods required using expensive reagents such as enzymes modified with PEG and antibodies, for suppressing reaction from lipoprotein other than HDL.
The precipitation method was also in the mainstream in the field of dry chemistry. However, a novel test piece for a dry process using the direct method has been developed in recent years and described in JP Patent No. 3686326. JP Patent Publication (Kokai) No. 2005-137360A (2005) has disclosed that the use of lipase derived from Candida rugosa improves selectivity. However, any of the test pieces cannot completely remove cholesterol in lipoprotein other than HDL.